Summary of the invention
The present invention is directed to the prior art deficiency, follow Traditional Chinese medical theory according to China, adopt the Chinese medicinal formulae treatment, reach the purpose that delays the ovary aging by " the kidney invigorating tonifying YIN, soothing the liver nourishing blood ".The invention provides anti-ovarian function that a kind of Semen Cuscutae Flos Carthami prescription forms effective Chinese medicine compound that fails, from the effective site aspect, by suitable method for extraction and purification and technology, extract as much as possible and keep wherein flavone component, to guarantee the drug effect of Chinese medicine compound, improve curative effect, reduce dose, active clear and definite, toxic and side effects is little.
The present invention seeks to realize by following technical scheme:
A kind of Chinese medicine flavone extract of anti-ovarian function decline is prepared from by the following method:
1) gets crude drug by following weight portion: Semen Cuscutae 100-400 part, Flos Carthami 7-32 part, mix according to quantity and doubly measure mixture after large polarity extracting solution stirs with 6-10, under static state extract, can adopt sonic oscillation method, heating and refluxing extraction method or countercurrent extraction method, make be dissolved in the large polarity extracting solution of active component of the raw material in the mixture more.Described large polarity extracting solution preferred water, methanol, ethanol, or their any mixed liquor; Particularly preferably be concentration 60-80%(percent by volume), pH value is the alcoholic solution of 5.6-6.3;
2) collect extracting solution, concentrating under reduced pressure is 0.8-1.3g/ml to density, with 3-6 defat of 30-60 ℃ of petroleum ether equal-volume extraction of boiling range, discard petroleum ether layer, get aqueous layer extracted and extract 3-6 time the rotary evaporation extract with the ethyl acetate equal-volume, obtain powder after the drying, for slightly carrying the flavone position.
3) adopt the HPD450 macroporous adsorbent resin, preferably slightly put forward the sample solution concentration loading of flavone/ml with 0.2g, use respectively 8 times of (comparing with the wet resin amount) distilled water flush away impurity, then use 70% ethanol elution purification total flavones, collect eluent, decompression recycling ethanol, concentrating under reduced pressure, the dry total flavone part that gets.
Above-mentioned Chinese medicine flavone extract can be made preparation for administration separately or with one or more pharmaceutically acceptable carrier combinations, such as injection, transfusion, drop pill, tablet, capsule, granule, oral liquid etc.
The various dosage forms of Chinese medicine flavone extract of the present invention can be prepared according to the method for knowing in the pharmaceutical field.Can contain for example active component of 0.05% ~ 90% weight with carrier combinations in these pharmaceutical formulations, the active component of weight between more common about 15% ~ 60%.
The present invention also provide a kind of Chinese medicine flavone extract for preparing anti-ovarian function decline of the present invention method, comprise the steps:
1) gets crude drug by following weight portion: Semen Cuscutae 100-400 part, Flos Carthami 7-32 part, mix according to quantity and doubly measure mixture after large polarity extracting solution stirs with 6-10, under static state use sonic oscillation method, heating and refluxing extraction method or countercurrent extraction method, make be dissolved in the large polarity extracting solution of active component of the raw material in the mixture more.Described large polarity extracting solution preferred water, methanol, ethanol, or their any mixed liquor; Particularly preferably be concentration 60-80%, pH value is the alcoholic solution of 5.6-6.3;
2) collect extracting solution, concentrating under reduced pressure is 0.8-1.3g/ml to density, with 3-6 defat of 30-60 ℃ of petroleum ether equal-volume extraction of boiling range, discard petroleum ether layer, get aqueous layer extracted and extract 3-6 time the rotary evaporation extract with the ethyl acetate equal-volume, obtain powder after the drying, for slightly carrying the flavone position.
3) adopt the HPD450 macroporous adsorbent resin, slightly put forward the sample solution concentration loading of flavone/ml with 0.2g, use respectively 8 times of (comparing with the wet resin amount) distilled water flush away impurity, then use 70% ethanol elution purification total flavones, collect eluent, decompression recycling ethanol, concentrating under reduced pressure, the dry total flavone part that gets.
The present invention also provides the application of a kind of above-mentioned Chinese medicine flavone extract in the anti-ovarian function decline of preparation medicine.
The Chinese medicine compound that Chinese medicine flavone extract of the present invention adopts is comprised of the Semen Cuscutae Flos Carthami, wherein, Semen Cuscutae is warm in nature, sweet in the mouth, energy nourishing the liver and kidney, controlling nocturnal emission with astringent drugs reducing urination, antiabortive, make eye bright, antidiarrheal is suffered from a deficiency of the kidney and is rushed down for impotence and seminal emission, dripping urination, enuresis frequent micturition, soreness of the waist and knees, blurred vision tinnitus, vaginal bleeding due to deficiency of the kidney during pregnancy, frequent fetal movement, spleen; Externally treating leucoderma.Flos Carthami is warm in nature, acrid in the mouth, and the latus rectum of invigorating blood circulation, eliminating stasis to stop pain are used for amenorrhea, dysmenorrhea, lochia, lump in the abdomen mass in the abdomen, traumatic injury, applicable various varicosis, peripheral neuritis, blood circulation is bad, lower limb numbness or the congestion such as livid purple.Semen Cuscutae is the medicine of YANG invigorating, and the kidney invigorating and essence nourishing is arranged, nourishing the liver to improve visual acuity, the effect of antidiarrheal, Flos Carthami tool promoting blood circulation to remove obstruction in the collateral, the merit of stasis-dispelling and pain-killing can reach the kidney invigorating and essence nourishing, nourishing YIN and benefiting blood with the Semen Cuscutae compatibility, Yin Yang balancing, the effect of QI and blood regulating is applicable to the card of the hepatic and renal YIN deficiency, is the medicine of the tonifying YIN of enriching blood.Semen Cuscutae contains flavonoid, resin glycoside, saccharide.Flos Carthami contains a large amount of flavone compounds, and other contains volatile oil, plant sterol, polysaccharide, aminoacid, fatty acid, fatty wet goods.Therefore for the anti-ovarian function that has Semen Cuscutae Flos Carthami prescription to form effective Chinese medicine compound that fails, from the effective site aspect, so by suitable method for extraction and purification and technology, extract as much as possible and keep wherein flavone component, to guarantee the drug effect of Chinese medicine compound, that Chinese medicine (compound recipe) improves curative effect, less dose, a kind of feasible method of increase technology content.
The present invention had both kept the comprehensive function of each medicinal ingredient in the compound recipe, was conducive to bring into play the pleiotropy of Chinese medicine, had removed again suck tissue and partial invalidity composition, had prolonged the storage life, had improved purity, had reduced dose, and the repeatability of quality control improves.
Above-mentioned Chinese medicine flavone extract confirms the anti-ovarian function decline of its tool function through pharmacological evaluation, the preferred polycystic ovarian syndrome of described ovary degradation symptoms, functional menoxenia, premature ovarian failure or menopausal syndrome.
The present invention overcomes the characteristics that existing capsule taking dose is large, store inconvenience, time weak point, by using suitable solvent extraction to separate to full side's raw medicinal material and filtering out drug effect higher part position, keep the medical material effective ingredient, remove suck tissue and partial invalidity composition, active obviously raising reduces dose, strengthens drug compliance, the repeatability of quality control improves, convenient storage and transportation.Method technique of the present invention is simple and safe, and the resulting composition drug effect is better than former side's medical material, and production cost is low, is suitable for suitability for industrialized production, and the compositions of preparing is that active component can be made into various pharmaceutical dosage forms, such as soft capsule, tablet, drop pill, controlled release agent etc.
The specific embodiment
Below with reference to specific embodiment the present invention is further elaborated, these examples only are used for illustration purpose, and are not used in the restriction scope of the invention.Laboratory animal of the present invention is available from Test Animal Centre, Academy of Military Medical Sciences, P.L.A (production licence number: SCXK (army) 2007-004).
The extraction purification of embodiment 1 Chinese medicine flavone extract of the present invention
Get double centner Semen Cuscutae and 7 kilograms of Flos Carthamis, add 1070 kilograms of pH value and be 5.6 60% soak with ethanol after 1 hour, refluxed 3 hours, 8 layers of filtered through gauze, medicinal residues are 5.6 60% alcohol reflux 2 hours with 428 kilograms of pH value; Concentrating under reduced pressure is 1.1g/ml to density, and with 5 defats of 30-60 ℃ of petroleum ether equal-volume extraction of boiling range, with ethyl acetate equal-volume extraction 3 times, the rotary evaporation extract obtains powder after the drying, for slightly carrying the flavone position.
Adopt the HPD450 macroporous adsorbent resin, with the sample solution concentration loading of 0.2g extract/ml, use respectively 8 times of (comparing with the wet resin amount) distilled water flush away impurity, then use 70% ethanol elution purification total flavones, collect eluent, decompression recycling ethanol, concentrating under reduced pressure, the dry total flavone part that gets.
The extraction purification of embodiment 2 Chinese medicine flavone extracts of the present invention
Get 250 kilograms of Semen Cuscutae and 20 kilograms of Flos Carthamis, pulverize the powder that obtains them through pulverizer respectively, the order numerical control is built in 80 orders.Be that 6 75% alcoholic solution mixes with above-mentioned powdery Semen Cuscutae Flos Carthami and 2160 kilograms of pH value, and in agitator, stir, then slowly flow into container through pipeline, tube wall at this pipeline is provided with supersonic generator, he carries out the sonic oscillation break process to the mixture of sluggish flow in the pipeline, processing time is 30 minutes, and the active component in the germinal cell fully is dissolved in the alcoholic solution.Mixture solution after the supersound process is filtered, remove residue, concentrating under reduced pressure is 1.0g/ml to density, with 3 defats of boiling range 30-60 ℃ of petroleum ether equal-volume extraction, with ethyl acetate equal-volume extraction 4 times, rotary evaporation extract, obtain powder after the drying, for slightly carrying the flavone position.
Adopt the HPD450 macroporous adsorbent resin, with the sample solution concentration loading of 0.2g extract/ml, use respectively 8 times of (comparing with the wet resin amount) distilled water flush away impurity, then use 70% ethanol elution purification total flavones, collect eluent, decompression recycling ethanol, concentrating under reduced pressure, the dry total flavone part that gets.
The extraction purification of embodiment 3 Chinese medicine flavone extracts of the present invention
Get 250 kilograms of Semen Cuscutae and 20 kilograms of Flos Carthamis, pulverize the powder that obtains them through pulverizer respectively, the order numerical control is built in 80 orders.Be 6 75% soak with ethanol after 1 hour with above-mentioned powdery Semen Cuscutae Flos Carthami and 2160 kilograms of pH value, refluxed 4 hours, 8 layers of filtered through gauze, medicinal residues are 6 75% alcohol reflux 3 hours with 1620 kilograms of pH value; Concentrating under reduced pressure is 0.8g/ml to density, and with 5 defats of 30-60 ℃ of petroleum ether equal-volume extraction of boiling range, with ethyl acetate equal-volume extraction 3 times, the rotary evaporation extract obtains powder after the drying, for slightly carrying the flavone position.
Adopt the HPD450 macroporous adsorbent resin, with the sample solution concentration loading of 0.2g extract/ml, use respectively 8 times of (comparing with the wet resin amount) distilled water flush away impurity, then use 70% ethanol elution purification total flavones, collect eluent, decompression recycling ethanol, concentrating under reduced pressure, the dry total flavone part that gets.
The extraction purification of embodiment 4 Chinese medicine flavone extracts of the present invention
Get 400 kilograms of Semen Cuscutae and 32 kilograms of Flos Carthamis, add 2592 kilograms of pH value and be 6.3 80% soak with ethanol after 1 hour, refluxed 3 hours, 8 layers of filtered through gauze, medicinal residues are 6.3 80% alcohol reflux 4 hours with 3456 kilograms of pH value; Concentrating under reduced pressure is 1.3g/ml to density, and with 5 defats of 30-60 ℃ of petroleum ether equal-volume extraction of boiling range, with ethyl acetate equal-volume extraction 3 times, the rotary evaporation extract obtains powder after the drying, for slightly carrying the flavone position.
Adopt the HPD450 macroporous adsorbent resin, with the sample solution concentration loading of 0.2g extract/ml, use respectively 8 times of (comparing with the wet resin amount) distilled water flush away impurity, then use 70% ethanol elution purification total flavones, collect eluent, decompression recycling ethanol, concentrating under reduced pressure, the dry total flavone part that gets.
Embodiment 5 sets up the Determination of Total Flavonoids method
1) reagent and instrument
Silon ethanol (analytical pure), methanol (analytical pure)
Rutin standard solution: take by weighing the 5.0mg rutin, add dissolve with methanol and be settled to 100mL, namely get 50 μ g/mL.
2) operational approach
Sample pretreating: take by weighing a certain amount of sample, add ethanol and be settled to 25mL, after shaking up, supersound extraction 20min places, and draws supernatant 1.0mL, in evaporating dish, adds the absorption of 1g Silon, flings to ethanol in water-bath, then changes chromatographic column over to.Wash with 20mL benzene first, benzene liquid discards, and then uses the methanol-eluted fractions flavone, is settled to 25mL.This liquid is measured absorption value in wavelength 286nm.Take rutin as standard substance, the bioassay standard curve is asked regression equation simultaneously, calculate general flavone content rutin standard curve in the sample: draw rutin standard solution 0,1.0,2.0,3.0,4.0,5.0mL in the 10mL color comparison tube, add methanol to scale, shake up, in wavelength 286nm colorimetric.Ask regression equation, calculate general flavone content in the sample.
Calculating and result represent (result of calculation keeps two position effective digitals)
In the formula: the content of total flavones in the X-sample, mg/100g; A-calculates to get flavone amount in the test solution, μ g by standard curve; The M-sample mass, g; V1-measures and uses volume of sample, mL; V2-sample standardize solution cumulative volume, mL.
Embodiment 6 slightly carries flavone purifying process conditional filtering
The simple flavone that adopts alcohol extracting method, hot water extraction or alkaline extraction all can't obtain high-load, the extracting solution of compound recipe or crude extract must could improve purity through after the separation and purification, purification process has recrystallization method, polyamide absorption column chromatography method and Flavonoids by Macroporous Adsorption Resin, also has in addition ultrafiltration, high performance liquid chromatography (HPLC) and blood capillary electrophoresis method etc.The industrial conventional Flavonoids by Macroporous Adsorption Resin that adopts.
1) material and reagent
HPD600, HPD480, HPD700, HPD750, HPD480 macroporous resin (Cangzhou, Hebei precious grace chemical industry company limited);
D101 macroporous resin (China Medicine University)
2) use macroporous adsorbent resin crude flavonoid powder is carried out purification
Study the Adsorption and desorption characteristics of macroporous adsorbent resin to flavone, therefrom filtered out the resin that is suitable for flavone of the present invention position purification, and used selected macroporous adsorbent resin that purification is carried out at the flavone position.
The pretreatment of resin
With wet method dress post behind the macroporous adsorbent resin usefulness soaked in absolute ethyl alcohol 24h; After resin installs,, add water to effluent and be not white in color till the muddiness, and be washed till without ethanol with same flow velocity with deionized water by resin bed with ethanol 2 BV/h; Then with 2 BV 0.5%HCl solution with 2.5 ml/min flow velocitys by resin bed, and soak behind the 2-4h water and be washed till the effluent pH value with same flow velocity and be neutral; Pass through resin bed with 2 BV 2%NaOH solution with the 2.5ml/min flow velocity at last, and after soaking 2-4 h, water is washed till effluent pH with same flow velocity and is neutral.
Different model resin technology index
The technical specification of several model macroporous adsorbent resins that we select sees table 1 for details.
Table 1 different model macroporous adsorbent resin technical specification
3 Staticadsorption experiment
Take by weighing respectively each each 1.0g of model resin (dry weight), put into the 250ml triangular flask, accurately add in the embodiment of the invention 1 and slightly carry flavone position extracting solution 100ml, place the water bath chader, be that 40 ℃, frequency of oscillation are under the 100Hz/min condition in temperature, vibration 12h is fully after the absorption, filter, get filtrate and survey its concentration.Be calculated as follows each resin adsorbance (Q, mg/g) at room temperature, determination data sees table 2 for details.
0r
In the formula: C
0Be the concentration (mg/ml) of adsorbing front flavone; C
rBe the concentration (mg/ml) of adsorbing rear flavone; V is sample solution volume (ml); W is resin quality (g).
Table 2 macroporous resin to the static adsorption Performance Ratio of flavone
As seen the static adsorbance size is arranged as: HPD700〉HPD750〉D101〉HPD480〉HPD450〉HPD600.
Associative list 1 and as seen from Table 2: nonpolar resin is higher than polar resin to the static adsorbance of this compound recipe flavone.The distinctive porous network structure in macroporous resin surface makes it have molecular sieving effect to Organic substance, and namely Organic substance can be diffused into by the aperture of resin the resin internal surface of hole and is adsorbed.The pore size of resin directly affects freely coming in and going out of different sized molecules, thereby makes resin have certain selectivity.Therefore, the absorbability of macroporous resin is relevant with molecular weight and the configuration of adsorbed material.
Static desorption experiment
Accurately take by weighing each the model resin after abundant saturated adsorption is slightly carried flavone, add respectively 30%, 50%, 70% and 90% each 30ml of alcoholic solution.Put into the water bath chader 12h that vibrates, filter, measure flavones content in the filtrate, calculate the desorption efficiency of each resin according to following formula, determination data sees table 18 for details.
Desorption efficiency (%)=C*V/Q*W*100
In the formula: C is the concentration (mg/ml) of flavone in the stripping liquid; V is the volume (ml) of eluent; Q is resin absorption amount (mg/g); W is the quality (g, weight in wet base) of used resin
Table 3 macroporous resin compares the static desorption performance of flavone
By above data analysis, visible alcoholic solution increases along with the increase of concentration of alcohol the desorption ability of flavone; And the alcoholic solution of same concentrations to the desorption ability that is adsorbed on the flavone on the resin is: HPD450〉HPD750〉HPD700〉D101〉HPD480〉HPD600.
Conclusion: by the macroporous resin of different model to the analysis of flavone static adsorption and desorption ability as seen, these six kinds of macroporous resins are more or less the same to the static adsorbance of flavone, but it is larger that desorption ability just differs, and only has HPD450 and HPD750 desorption ability stronger, can reach more than 80%.
The dynamic adsorption experiment
During the dress post, remain that resin is immersed in the distilled water, avoid Bubble formation on the impact of flavone separation and purification.After resin is added to predetermined column volume, repeatedly cross post with distilled water again, the resin in the post is fully compressed.The static placement of the resin column that installs is spent the night, and makes the resin in the post continue to compress until could use after fully settling out.During loading post top distilled water put to after the resin face flushes substantially, slowly add the sample solution of purification to be separated along tube wall from post top.Should note not communications centre pine top layer resin and make it floating and just rise.Under post, emit a little liquid, substantially flush with resin surface to the sample liquid level.Then add eluant, flow speed control is at 3-4ml/min, and the Fractional Collections eluent is measured flavone concentration wherein, draws elution curve.
Optimize the resin of HPD450 and two kinds of models of HPD750 by static adsorption and static desorption experiment, we just carry out the dynamic adsorption experiment relatively to these two kinds of model resins.As eluant, the flavone that is adsorbed on HPD450 and the HPD750 resin is carried out eluting with 90% alcoholic solution, every interval 30ml collects eluent, measures the concentration of flavone in the different eluant components, and concrete data see Table 4.Can find out, the elution curve peak shape of HPD450 resin is sharp-pointed, conditions of streaking is not obvious, show with 90% ethanol can make the flavone that is adsorbed on the HPD450 resin fast as eluant and the concentrated area elutes, further shown the desorption performance that the HPD450 resin is good to flavone.
Two kinds of macroporous resins of table 4 are to the dynamic desorption of flavone
The investigation of 6 sample solution concentration
Get 3 parts of each 10g of resin HPD450 that handle well, be respectively charged in the post of 2cm * 20cm, get a certain amount of flavone position of slightly carrying and so that not commensurability pure water is ultrasonic it is dissolved fully, be made into concentration and be respectively 0.2g extract/ml, 0.1g extract/ml, the sample liquid of 0.05g extract/ml, respectively complete loading, collect effluent, with the flavone sample liquid of not adsorbing between 20ml distilled water flushing resin slit, collect simultaneously, HPLC measures the content of its flavone, calculate saturated extent of adsorption, the results are shown in Table 5.
Table 5 sample solution concentration is investigated the result
Sample solution concentration (the g extract/ml) | 0.2 | 0.1 | 0.05 |
The sample solution peak area | 4990524.5 | 201393 16 | 17558314 |
Flavone amount (g) in the sample solution | 0.52737939 | 0.42564905 | 0.37109899 |
The effluent peak area | 4607545.5 | 5902886 | 8508222 |
Flavone amount (g) in the effluent | 0.09738153 | 0.12475885 | 0.08991161 |
Resin heavy (g) | 10.005 | 10.006 | 10.011 |
Flavone adsorbance (mg/g resin) | 42.98 | 30.07 | 28.09 |
Sample solution concentration is larger, adsorption effect is better, but just be difficult to dissolving when in experimentation, finding preparation 0.2g extract/ml sample liquid, need long ultrasonic could the dissolving, the concentration that increases again sample solution just can not make it all dissolve, so we think that the sample solution concentration of 0.2g extract/ml is optium concentration (wherein the concentration of flavone is 15.33mg/ml).
. the investigation of absorption flow velocity
Get runic flavone aqueous solution (concentration of flavone is 15.33mg/ml) 34.4ml, be splined on respectively 6 10gHPD450 macroporous resins, respectively with 2,4,5,6, the flow velocity of 8ml/min carries out dynamic adsorption, take the content of flavone as index, measure the concentration after adsorbing, calculate saturated extent of adsorption, the results are shown in Table 6.
The investigation of table 6 absorption flow velocity
Adsorption rate (ml/min) | Loading concentration (mg/ml) | Concentration after the loading (mg/ml) | Loading volume (ml) | Saturated extent of adsorption (mg/g) |
2 | 15.331 | 5.616 | 34.4 | 33.392 |
4 | 15.331 | 4.933 | 34.4 | 35.741 |
5 | 15.331 | 4.552 | 34.4 | 37.036 |
6 | 15.331 | 6.280 | 34.4 | 31.102 |
8 | 15.331 | 12.055 | 34.4 | 11.259 |
The result shows that when flow velocity was 2,4,5ml/min, the effect difference of absorption was very little, and is increased to 6ml/min when above, and adsorbance begins to descend with the increase of flow velocity, so this experiment is finally take 5ml/min as adsorbing flow velocity.
The investigation of washing amount
In the purification by macroporous resin process, owing in washing desaccharide process, the loss of flavone is arranged, so need to investigate it.Get the leakage that Exocarpium Citri Grandis aqueous solution (concentration of flavone is 15.33mg/ml) 15ml(assurance does not almost have loading to cause), be splined on the HPD450(10g of the 2cm * 20cm that handles well) on, fully use the deionized water eluting behind the absorption 12h, every 10ml is a unit, utilize the alpha-Naphthol concentrated sulphuric acid to carry out chromogenic reaction, measure simultaneously the content of flavone in the effluent.The results are shown in Table 7.
The investigation of table 7 washing amount
Washing amount (ml) | Alpha-Naphthol concentrated sulphuric acid chromogenic reaction color | Reaction result | The concentration of flavone (mg/ml) in the effluent |
0~10 | The darkviolet colour circle | Reacting positive- | |
10~20 | The darkviolet colour circle | Reacting positive | 0.0013 |
20~30 | Than the darkviolet colour circle | Reacting positive | 0.0120 |
30~40 | The purple colour circle | Reacting positive | 0.0124 |
40~50 | The purple colour circle | Reacting positive | 0.0339 |
50~60 | The lilac colour circle | Reacting positive | 0.0414 |
60~70 | Without colour circle | Reaction negative | 0.0540 |
70~80 | Without colour circle | Reaction negative | 0.0940 |
Sucrose solution | The darkviolet colour circle | Reacting positive | |
Pure water | Without colour circle | Reaction negative | |
The result shows, sugared reaction negative after the 70ml washing, flavone begins along with the increase of washing amount to run off, in order to guarantee sugared reaction negative and to guarantee in the less situation of flavone loss, some water-solubility impurities of removing more, the water elution amount is decided to be 80ml, i.e. 8 times of applied sample amount volumes, and the loss of flavone is 1.08% in this water-washing process.
So final definite: as to adopt the HPD450 macroporous adsorbent resin, sample solution concentration loading with 0.2g extract/ml, use respectively 8 times of (comparing with the wet resin amount) distilled water flush away impurity, then use 70% ethanol elution purification total flavones, collect eluent, decompression recycling ethanol, concentrating under reduced pressure, the dry total flavone part that gets.
The embodiment 7 different chemical parts that extract are on the impact of the gonad granulocyte activity that exsomatizes
Sample preparation: sample 1: by the total flavone part behind the purification that obtains in the embodiment of the invention 1; Sample 2: by the crude flavonoid powder part that obtains before the embodiment of the invention 1 center pillar chromatographic step.
Animal experiment method: get 22-27 age in days female rats, the cervical vertebra dislocation method is put to death behind the conventional 48h of raising, the aseptic bilateral ovaries of winning rat, granular cell is discharged the ovary fragmentation, contain with 2mL in the DMEM-F12 culture fluid of penicillin and streptomycin and dilute, and gently piping and druming, make it be dispersed into individual cells.Centrifugal filtration collecting granules cell.(1) mtt assay: with the finite concentration cell suspension inoculation in 24 orifice plates, every hole 80 μ l, every hole adds 120 μ l culture fluid again and adjusts cell density to 200 μ l/ hole, establishes 6 parallel holes for every group.Be placed on 5%CO
2Cultivate 24h in the incubator.Get CO
2Cultivated the granular cell suspension of 24h in the incubator, every hole successively adds modeling concentration Caddy (Cleary) and 1mgmL
-1Different samples, parallel setting Normal group (normal saline) and model group (Caddy (Cleary) and normal saline).In every hole, add MTT solution (5mgmL behind the cellar culture 20h
-1) 20 μ L continuation cultivation 4h, every hole adds three liquid of 80 μ L, and incubated overnight is selected the 570nm wavelength, measures each hole absorbance at enzyme-linked immunosorbent assay instrument, the results are shown in Table 8.(2) radioimmunology: with the finite concentration cell suspension inoculation in 24 orifice plates, every hole 80 μ l, every hole adds 120 μ l culture fluid again and adjusts cell density to 200 μ l/ hole, establishes 6 parallel holes for every group.Be placed on 5%CO
2Cultivate 24h in the incubator.Get CO
2Cultivated the granular cell suspension of 24h in the incubator, every hole successively adds modeling concentration Caddy (Cleary) and 1mgmL
-1Different samples, parallel setting Normal group (normal saline) and model group (Caddy (Cleary) and normal saline).Be placed on 5%CO
2Cultivate 24h in the incubator.Its cell conditioned medium liquid of centrifuging and taking is measured E with radioimmunological kit
2, P content, the results are shown in Table 8.(3) flow cytometry: after obtaining gonad granulocyte with method, the finite concentration cell culture in culture bottle, is successively added modeling concentration Caddy (Cleary) and 1mgmL
-1In different samples and the culture bottle, parallel setting Normal group (normal saline) and model group (Caddy (Cleary) and normal saline), digestion obtains cell suspension and carries out flow cytometer and detect (see figure 1) after 24 hours, analyzes the relation with apoptosis of administration and gonad granulocyte.
Mtt assay and radioimmunology as a result display model group and Normal group all have significant difference (p<0.01) on cell-proliferation activity and estrogen and progestogen secretion capacity, visible modeling success.And under the same dose, sample 1 is suitable in the cell-proliferation activity effect with sample 2, but sample 1 estrogen and progestogen secretion capacity is better than sample 2, but all with model group significant difference (all p<0.01) is arranged.
Table 8 is different extract chemical part on the impact of the gonad granulocyte activity that exsomatizes (
N=6)
Group | Positive controls | Sample 1 | Sample 2 | Model group | Normal group |
MTT trap (A) | 0.435±0.091
** | 0.415±0.024
** | 0.414±0.061
** | 0.233±0.064
ΔΔ | 0.465±0.093 |
Estrogen (pmol/L) | 83.89±5.33
** | 81.29±1.86
** | 76.36±2.51
** | 36.19±1.98
ΔΔ | 101.85±6.38 |
Progestogen (nmol/L) | 0.38±0.06 | 0.59±0.02
** | 0.50±0.04
* | 0.36±0.03
ΔΔ | 0.69±0.07 |
*P<0.05,
*P<0.01vs model group.
ΔP<0.05,
△ △P<0.01vs Normal group
The flow cytometry result shows: in the accompanying drawing 1, UL represents dead cell, and UR represents the apoptosis in late period, and LL represents living cells, and LR represents early apoptosis, and UR+LR is the apoptosis total value.Figure 1A (Normal group) apoptosis value is 7.30, Figure 1B (model group) apoptosis value is 56.42, Fig. 1 C(positive controls) the apoptosis value is 27.02,1 group in Fig. 1 D(sample) the apoptosis value is 26.30,2 groups in Fig. 1 E(sample) the apoptosis value is 33.64, suitable to the anti-apoptotic effect of gonad granulocyte with positive drug (estradiol valerate) according to the visible sample of numerical value 1, be better than sample 2.
The anti-apoptotic effect of comprehensive gonad granulocyte cultivation effect, estrogen and progestogen secretion level and gonad granulocyte, every action activity that visible sample 1 is the flavone position behind the purification is all good, and purifying process is necessary.
Embodiment 8 Chinese medicine flavone extracts of the present invention are on the impact of Model of Polycystic Ovarian Syndrome rat
24d age teenage SD female rats, body weight 30-40g, 0.5mg18-methylnorethindron day 1 subcutaneous injection, at 27d during age, add with HCG 1.5IU day 2 subcutaneous injections, inject altogether 21d.After the modeling success, be divided at random the flavone position behind model group, the sample 1-4(purification, preparation is respectively with embodiment of the invention 1-4) group and positive drug group (metformin 0.018g/d), every group each 10, other gets 10 of 45d SD in age female rats and is Normal group, and every day, gavage was 1 time, Normal group and model group give the isometric(al) normal saline, behind the successive administration 15 days, get blood examination after the last administration and survey FSH, LH, T concentration in the serum, the results are shown in Table 9.Display model group and Normal group all have significant difference (p<0.01) as a result, gavage positive drug, the present invention's (high low dosage) after 15 days FSH, LH, T level the difference of different significances is all arranged from model group.
The comparison of FSH, LH, T concentration in of the present invention group of table 9 and the matched group serum (
N=10)
*P<0.05,
*P<0.01vs model group.
ΔP<0.05,
△ △P<0.01vs Normal group
Embodiment 9 Chinese medicine flavone extracts of the present invention are on the impact of premature ovarian failure sex hormone level in rats
Adopt 9d SD in age female rats, continuously 14 days cyclophosphamide 8mg/(kgd of lumbar injection) modeling.After the modeling success, be divided at random the flavone position behind model group, the sample 1-4(purification, preparation is respectively with embodiment of the invention 1-4) group and positive drug group (premarin 0.075mg/kg), every group each 10, other gets 10 of 23d SD in age female rats and is Normal group, and every day, gavage was 1 time, and Normal group and model group give the isometric(al) normal saline, behind the successive administration 35 days, get blood examination after the last administration and survey FSH, E in the serum
2Concentration the results are shown in Table 10.Display model group and Normal group all have significant difference (p<0.01) as a result, gavage positive drug, the present invention's (high low dosage) FSH, E after 35 days
2Level all has the difference of different significances from model group.
FSH, E in of the present invention group of table 10 and the matched group serum
2The comparison of concentration (
N=10)
*P<0.05,
*P<0.01vs model group.
ΔP<0.05,
△ △P<0.01vs Normal group.